A major effort of our laboratory this past year was directed at characterizing the trafficking of key proteins in neurons of the central nervous system and hair cells of the inner ear. In neurons, much of our research has focused on NMDA receptors and their associated proteins. Since the NMDA receptor performs a critical function at the synapse and is a key player in synaptic plasticity, it is important to understand how this receptor is delivered to the synapse and how the number and composition of receptors at the synapse are regulated. In addition to being at the synapse, some NMDA receptors are extrasynaptic where they may have functions distinct from those at the synapse. We have also carried out research on the trafficking of PMCA2, which is abundant in the stereocilia of hair cells and is important for the removal of calcium from the stereocilia. Mutations in this protein cause deafness. [unreadable] [unreadable] This past year we published a study on LGN, the mammalian homologue of Drosophila Partner of Inscuteable (Pins), and its relationship to the trafficking of NMDA receptors in neurons. This project was presented in detail in the Annual Report of 2005. [unreadable] [unreadable] We have been studying the role of a PDZ protein, GIPC (GAIP interacting protein, C-terminus), in the trafficking of the NMDA receptor. GIPC was identified as an NR2 interacting protein through a yeast two-hybrid screen. Expressed in heterologous cells, it associates with the NMDA receptor both on the cell surface and in intracellular organelles. In cultured neurons, over-expression of GIPC increases the number of NMDA receptors on the cell surface, while expression of a dominant negative construct or an RNAi decreases surface label. Our results show that GIPC stabilizes NMDA receptors on the cell surface. Using an internalization assay, GIPC over-expression decreases internalization. In dendrites, it is associated with a subpopulation (about 20%) of surface NMDA receptors. GIPC is widely distributed in neurons. Using both light and electron microscopy in cultures and intact tissue, GIPC is abundant near the synapse, but is not particularly enriched at the postsynaptic density. Subfractionation studies support these findings. Our current interpretation of these results is that GIPC has a preference for extrasynaptic NMDA receptors, and it may be stabilizing this population on the cell surface.[unreadable] [unreadable] This past year we completed a study on PMCA2 in stereocilia of hairs cells of the inner ear, resulting in two publications. A number of proteins, essential to the function of the hair cell, are located on the stereocilia, but little is known about their delivery and removal. Endo- and exocytotic vesicles are not found within the cytoplasm of the stereocilia indicating that addition and removal must occur at the base of the stereocilium. We chose PMCA2 as a model protein due to its relatively high abundance in stereocilia. We developed an antibody to its extracellular domain of PMCA2 and made a GFP-tagged construct of PMCA2. Our results on the removal of PMCA2 showed that it has a half-life in the order of 5-7 hours in the stereocilia, and that it exists in two pools, a high mobility pool and a more stationary pool. While PMCA2 is found only in the stereocilia of hair cells, a similar molecule, PMCA1, is found in the cell body. Furthermore, various splice variants of these two isoforms are also present in hair cells. We characterized the domains of PMCA1 and PMCA2 that are required for their specific targeting in hair cells by constructing various mutant constructs and transfecting them into cultured hair cells. We find that a large cytoplasmic loop on PMCA2 controls its specific delivery to the apical membrane (stereocilia). While no specific motif within this loop was identified, we found that the size of the loop was a critical factor in the targeting. Basolateral (cell body) targeting of PMCA1 depends on its cytoplasmic C-terminus.[unreadable] [unreadable] This past year we published the first study of a new family of proteins, SALMs (Synaptic Adhesion-like Molecules) that we identified through yeast two-hybrid screening using SAP97 as bait. This family consists of five members (we initially identified 4, and another group identified a 5th member), and all have PDZ-binding domains except SALM4 and SALM5. They have a single transmembrane domain and their extracellular domains contain leucine-rich motifs, an Ig domain and a FNIII domain. Over-expression of SALM1 enhances neurite outgrowth of cultured hippocampal neurons. We also find the over-expression clusters PDZ proteins as well as NMDA receptors in cultured neurons. Its association with NMDA receptors in neurons appears to depend on interactions of both molecules with PDZ proteins, such as PSD-95. However, we have also found that SALM1 interacts directly with the NR1 subunit of the NMDA receptors through their extracellular domains, raising the interesting possibility that this interaction is important to the stabilization of one or both of these proteins on the cell surface. This past year we carried out a study to determine if SALMs form heteromeric and/or homomeric complexes. When expressed in heterologous cells, we find that SALMs1-4 can assemble in all combinations, and co-immunoprecipitation studies on brain show that the SALMs are associated with each other. Through deletion analyses, our results suggest that SALMs dimerize associate their Ig and FNIII domains. Our initial studies on neurite outgrowth focused on SALM1. We have now studied SALMs2-4 and find that they also promote neurite outgrowth when overexpressed in cultured hippocampal neurons. We are investigating if both axon and dendrite growth are increased as well as characterizing other properties of the neurite outgrowth properties of the SALMs. To better understand the function of the SALMs, we carried out a distribution study using antibodies that we made to the four subunits. These results show a wide distribution of the SALMs. They were both pre- and postsynaptic, and present at both excitatory and inhibitory synapses. Although they are present throughout the brain, the amounts of the various individual SALMs vary widely.